Molecular Typing and antimicrobial sensitivity of Mycobacteria isolated from Yemeni Tuberculous Patients

Abstract

The aim of the present study was to characterize Mycobacteria isolated from Yemeni pulmonary TB patients to the species level and determine antibiotic sensitivity and resistance of isolated strains to routine antituberculosis drugs. One hundred seventy early morning sputum samples were collected from the Yemeni pulmonary TB patients referred to the National Tuberculosis Institute (NTI) in Sana’a – Yemen. Out of the 170 of Pulmonary TB cases 158 (93%) were new cases and 12 (7%) were old cases. Samples were processed by the Ziehel-Nelseen stain then the positive samples were inoculated in the special Ogawa’s TB medium and cultivated at 37◦C for 4-6 weeks. After successful cultivation the isolates were tested for sensitivity against the routine antibiotics treatment in Lowenstain-Jenseen medium. Few colonies (1 – 2 colonies) from positive inoculation L-J culture media were taken from the TB cultures and processed with the Phenol- Chloroform heating method to lyse the cell wall of the Mycobacteria and extract the DNA. All those procedures were done in the research microbiology laboratory in the NTI Sana’a Yemen. The extracted DNA were isolated and preserved in special microcenterfiuge tubes and transported to the Institute of Endemic Diseases (IEND), Khartoum University – Sudan for further molecular analysis because molecular techniques are not available till now in Yemen. 15 In the (INED) firstly small portion of isolated DNA were run in the Gel-Electrophoresis to be insured from the availability of DNA, then DNA samples were processed by PCR machine to detect and amplify the Mycobacteria DNA using specific primers complementary to the rpoB gene on the Mycobacteria DNA . After successful amplification of the target sequence of the DNA the PCR product were digested with specific restriction Enzyme by technique called Restriction Fragment Length Polymorphism (RFLP) then the digested DNA was electrophoresed in 2% agarose gel. DNA from All drug resistance isolates were amplified again separately and sent to the Korean Tuberculosis Institute (KTI) for doing DNA sequencing analysis. Results of our study showed that TB was present in all Yemen area and the highest rates of incidence were rerecorded in Hajah (18%), Alhodidh (14%), and Almaharh (12%) which are characterized by its hot weather and rural crowded provinces. Regarding gender our findings showed that the incidence of disease was high (69%) among males than females (31%) therefore the difference was significant. Infection was noted in patients at any age group but the disease was high (39%) among patients whose age ranged between 20 – 30 years. The antibiotics sensitivity results showed that; (91.2%) were sensitive to the four antibiotics: Isoniazid, Rifampicin, Streptomycin, and Ethambutol; but 15 (8.8%) were resistant to one or Multi drug resistant (MDR). Out of 15 resistant isolates 5/15 (33.3%) were resistant to only one type 16 of antibiotic "mono-resistant" whereas 10/15 (66.7%) were multi drug resistant MDR-TB. The highest percent of MDR-TB isolates were resistant to Isoniazed 14/15 (93%) followed by Rifampicin 8/15 (53%), Ethambutol 6/15 (40%) and Streptomycin 5/15 33%. According to the history of disease 9/15 (60%) of those MDR-TB were new cases and 6/15 (40%) were old cases. The molecular PCR thermocyclear results showed that the DNA sequence of rpoB gene was amplified successfully using (TB-MF and MR) primers. But the RFLP results showed the PCR-RFLP using (Hind11) restriction enzyme gave very clear, more reliable, and easier in the interpretation of the result than the (Msp1) enzyme. Out of 120 samples 118 (98.3%) were Mycobacterium tuberculosis complex (MTC) and two samples (1.3%) were Mycobacterium other than tuberculosis complex (MOTT). Our findings showed that MOTT strains were not sensitive to all antibiotics. Both MOTT strains isolates were resistant to three types of antibiotics TB treatment; Isoniazid, Rifampicin, and Streptomycin and sensitive only to Ethambutol. In addition the DNA sequencing analysis results showed that the alignment of nucleic acid of DNA in MOTT is different from that of MTC. Also DNA sequencing alignment shows that one isolate of MDR-TB which was collected from a TB patient comes from Somalia and harbored various nucleic acid sequence which suggested that this strain may be M.africanum.